Certain types of cellular cysteine proteases may play an important role in modulating the activity of G-protein coupled receptors. The activity of this class of enzymes is closely regulated by cytoplasmic and nuclear inhibitors. The effects of ethyl alcohol exposure on cysteine protease activity has been studied in cell culture utilizing a PC12 cell line. We have previously established that exposure of PC12 cells to ethyl alcohol for 96 hours results in a decrease in calcium-stimulated protease activity. We developed assays to measure GSH, GSSG, and NO and we have determined that both GSH and GSSG are important modulators of calpain activity. GSH is an uncompetitive inhibitor while GSSG is a competitive inhibitor of calpains, with inhibition constants well within range of concentrations of GSH and GSSG found in cells. We have also determined that nitric oxide also strongly inhibits protease activity. These results may relate to the previous reports that both ischemia and oxidative stress increase calpain activity in several mammalian cell types, and both processes are known to decrease both GSH and GSSG. We are now examining how alcohol exposure alters GSH, GSSG and NO as possible mechanisms for alcohol induced alterations in protease activity.